The decarboxylation of malic acid to lactic acid by certain species of lactic acid bacteria in the production of wine has been recognized for a long time. As malic acid is one of the most common organic acids in grapes (and other fruit), its decarboxylation to lactic acid results in a significant reduction of the acidity of the wine; this is particularly important for wines produced from grapes grown in cool areas as these have a high natural acidity. Another advantage of the conversion of malic acid to lactic acid (which in the literature and in the following description is often termed malolactic fermentation, for which reason the lactic acid bacteria capable of converting malic acid to lactic acid are termed malolactic bacteria in the present context) is that no further microbial fermentations may take place in the wine so that it becomes bacteriologically stable. Furthermore malolactic fermentation may exert some influence on the flavour of the wine.
In traditional vinification the decarboxylation process has been allowed to occur spontaneously as a result of the growth of an indigenous flora of lactic acid bacteria originating from the vines and grape skins and also often surviving on winery equipment, especially wooden casks or other equipment made of wood, from one season to the next, these bacteria decarboxylating malic acid to produce lactic acid. When occurring in this fashion, malolactic fermentation is often delayed and may take place several months after the alcoholic fermentation. The initial number of bacteria is often quite small and the environment of the wine is frequently rather hostile to the growth of these bacteria due to the content of ethanol and sulphur dioxide in the wine, as well as its low pH and nutrient concentration. The long lag phase of the malolactic bacteria during which the wine is biologically unstable may result in the growth of bacteria which produce volatile acidity and hence spoilage of the wine. Apart from this certain malolactic bacteria spontaneously growing in the wine may produce certain compounds, e.g. diacetyl and acetoin, that tend to give rise to "off" flavours or odours in the wine.
Even so, it is common practice to stimulate malolactic fermentation by reducing the content of sulphur dioxide, delaying the removal of the lees, increasing the temperature of the wine or ensuring a pH of more than 3.4. These conditions, however, also encourage the growth of other microorganisms in the wine, thus increasing the likelihood of wine spoilage and this approach therefore requires extremely careful supervision of the decarboxylation process.
Furthermore, the self-inoculation of the wine by indigenous bacteria is difficult, not to say impossible, to control, and its occurrence has become even more unpredictable as winery hygiene has improved stainless steel tanks having in many cases replaced wooden casks etc. Such measures serve to ensure a uniform quality of the wine and reduce the risk of spoilage. However, they also reduce the chance of spontaneous conversion of malic acid taking place in the wine. For this reason and because the winemaker will often prefer to exercise a greater degree of control over the decarboxylation process strains of malolactic bacteria have recently been isolated and cultivated with the purpose of adding them to wine or must in order to make sure that malolactic fermentation will take place.
In the previously known methods of inducing decarboxylation of malic acid by means of a selected strain of a malolactic bacterium, a culture of the malolactic bacterium which has been reactivated (i.e. a frozen or lyophilized culture which is adapted and/or propagated in a medium containing nutrients, must or wine) prior to use is added to fermented wine (the most usual approach) or, less frequently, to must before the alcoholic fermentation. The reactivation takes at least 24 hours and usually up to several days and requires special facilities in the winery for the propagation of the cultures. Furthermore, the bacteria may be susceptible to phage attack during the propagation period, or the culture may become contaminated with other microorganisms which are undesirable in wine, for which reasons the use of reactivated cultures may not always result in a satisfactory malolactic fermentation in the wine.
Although reactivation or propagation of the malolactic cultures is generally recommended in the literature, attempts have been made to inoculate wine directly with rehydrated lyophilized malolactic bacteria (R. B. Beelman and G. R. Duke. "The development and utilization of freeze-dried malolactic cultures for inoculation of wine" in Malolactic Fermentation, ed. T. H. Lee, Proceedings of a seminar organized by the Australian Society of Viticulture and Oenology Inc. and held 16th August 1984 in Melbourne, Victoria, pp. 53-63). However, this resulted in a quite lengthy lag phase (where no malic acid was converted) before commencement of malolactic fermentation, and reactivation of the cultures was therefore recommended. Experiments have also been carried out where rehydrated malolactic bacteria were added directly to grape must (R. B. Beelman and R. E. Kunkee, "Inducing simultaneous malolactic-alcoholic fermentation in red table wines" supra, pp 97-112), resulting in induction of malolactic fermentation. At economically feasible inoculation levels, the conversion of malic acid however, was not complete at a desirably fast rate, taking about 14-28 days, and propagation of the lyophilized cultures prior to inoculation was therefore recommended.
The bacterium most usually indicated for the deliberate induction of malolactic fermentation is Leuconostoc oenos, originally isolated from wine Leuc. oenos. however, often requires adaptation to the particular wine to which it is added in order to grow. Furthermore, when it is added directly to wine or must without any prior reactivation, it shows a quite lengthy lag phase of up to several weeks before it grows to a population which is sufficiently numerous to be active in decarboxylating the malic acid present in the wine resulting in a greater risk of wine spoilage due to contamination with organisms producing volatile acidity. Furthermore, Leuc. oenos is heterofermentative (i.e capable of producing volatile acidity so that it can only be used safely after the alcoholic fermentation is complete, thus retarding the malolactic fermentation. Also, malolactic fermentation by Leuc. oenos is inhibited at pH values below 3.2 which is less convenient as malolactic fermentation is most desirable in acidic wines.
The only other malolactic bacterium indicated for deliberate induction of malolactic fermentation seems to be Lactobacillus hilgardii isolated from wine. The strain is, however, described as heterofermentative, thus giving rise to the same limitations in its use as Leuc. oenos, and is, indeed, only indicated for use in admixture with a Leuc. oenos strain in a composition intended for reactivation before addition to fermented wine.
In order to ensure a high efficiency of the decarboxylation of malic acid and an easy inoculation procedure, it is therefore required to use a malolactic organism which possesses a high activity which is safer in that decarboxylation takes place substantially without the formation of volatile acids, which is suited for direct inoculation without showing any lag phase and which tolerates a low pH.